NEUTROPHILSNEUTROPHILS

Dr. P. ConcepcionDr. P. ConcepcionFellow-in-Training, Allergy & ImmunologyFellow-in-Training, Allergy & Immunology

Philippine General HospitalPhilippine General Hospital

April 25, 2014April 25, 2014

Sources:Sources: Adkinson et’al; Middleton’s Allergy Principle and Adkinson et’al; Middleton’s Allergy Principle and

Practice; 8Practice; 8 thth ed; Vol 1; Elsevier Saunders; 2014 ed; Vol 1; Elsevier Saunders; 2014 Abbas et’al; Cellular and Molecular Immunology; 7Abbas et’al; Cellular and Molecular Immunology; 7 thth ed; ed;

Elsevier Saunders; 2012Elsevier Saunders; 2012 Journals:Journals:

--Bruce K. et al: Bruce K. et al: Immunomodulatory Activity and Effectiveness of Immunomodulatory Activity and Effectiveness of

Macrolides in Chronic Airway DiseaseMacrolides in Chronic Airway Disease

- - Soichiro et’al: Soichiro et’al: Mechanisms of Action and Clinical Application of Mechanisms of Action and Clinical Application of

Macrolides as Immunomodulatory Medications, Macrolides as Immunomodulatory Medications, Clin. Microbiol. Rev. Clin. Microbiol. Rev. 2010, 2010, Journal ASM. OrgJournal ASM. Org

- - Evangelos, J., Evangelos, J., Macrolides beyond the conventional antimicrobials:Macrolides beyond the conventional antimicrobials:

a class of potent immunomodulators, a class of potent immunomodulators, International Journal of Antimicrobial International Journal of Antimicrobial Agents 31 (2008) 12–20Agents 31 (2008) 12–20

- - Tauber, S., Tauber, S., Immunomodulatory Properties of Antibiotics, Immunomodulatory Properties of Antibiotics, Current Current Molecular Pharmacology, 2008, 1, Bentham Science Publishers Ltd.Molecular Pharmacology, 2008, 1, Bentham Science Publishers Ltd.

Presentation Outline:Presentation Outline:

Introduction of Neutrophils’ BiologyIntroduction of Neutrophils’ BiologyThe Birth & the MorphologyThe Birth & the MorphologyDiscuss each step of its Key Discuss each step of its Key

Role in innate immune Role in innate immune defenses defenses Neutrophil Clearance and DeathNeutrophil Clearance and DeathNeutrophil-Associated DiseasesNeutrophil-Associated DiseasesDiscuss some Immunomodulators in contextDiscuss some Immunomodulators in context

• GranulocytesGranulocytes• Polymorphonuclear (PMNs)Polymorphonuclear (PMNs)• Polymorphonuclear Polymorphonuclear

leukocytes (PMNLs)leukocytes (PMNLs)• Poly’sPoly’s• SegmentersSegmenters

Neutrophil

Important role in inflammatory Important role in inflammatory responses responses

Comprise 50–75% of circulating Comprise 50–75% of circulating leukocytes in humansleukocytes in humans

First circulating cells to migrate to the First circulating cells to migrate to the site of infectionsite of infection

Uncontrolled activation causes tissue Uncontrolled activation causes tissue damage--- contribute to the pathogenesis of damage--- contribute to the pathogenesis of

chronic inflammation (eg sinuses and chronic inflammation (eg sinuses and respiratory tract)respiratory tract)

Introduction:Introduction:

• The The neutrophilsneutrophils are specialized are specialized for the phagocytosis and for the phagocytosis and destruction of micro-destruction of micro-organisms and damaged or organisms and damaged or necrotic tissues.necrotic tissues.

Introduction:Introduction:

6 Subtypes:6 Subtypes:

1. myeloblast1. myeloblast

2. promyelocyte----- primary (azurophilic) granules2. promyelocyte----- primary (azurophilic) granules

3. myelocytes---- secondary (specific) granules3. myelocytes---- secondary (specific) granules

4. metamyelocytes--- tertiary (gelatinase) granules4. metamyelocytes--- tertiary (gelatinase) granules

5. bands cells5. bands cells

6. mature neutrophils6. mature neutrophils

• Continuously generated from the BM (1-2 x 1011 cells/day --- amplified in times of stress

(eg infection)• Neutrophil maturation in the bone marrow

takes approximately 10–15 days,

Neutrophil – electron micrographNeutrophil – electron micrograph

Peter Newburger, MDUniversity of Massachusetts Medical School

A.A. An abundance of An abundance of granulesgranules

B.B. Multi-lobed nucleusMulti-lobed nucleus

C.C. Prominent Prominent cytoskeleton for cytoskeleton for locomotion and locomotion and chemotactic functionschemotactic functions

1. microfilaments1. microfilaments

2. microtubules2. microtubules

3. intermediate 3. intermediate filamentsfilaments

Morphology:Morphology:

CirculationCirculation

Most abundant WBC in the bloodMost abundant WBC in the bloodThe half life = 4-10 hoursThe half life = 4-10 hours11 (ave: 6hours (ave: 6hours2)2)

Can migrate to the site of infectionCan migrate to the site of infection If not--- it undergoes Apoptosis and If not--- it undergoes Apoptosis and

phagocytosed by resident macrophagesphagocytosed by resident macrophages

1. Adkinson et’al; Middleton’s Allergy Principle and Practice; 8th ed; Vol 1; 2014 2. Abbas et’al; Cellular and Molecular Immunology; 7th ed;; 2012

CirculationCirculation

Peripheral blood neutrophils are Peripheral blood neutrophils are divided between;divided between; A Circulating pool- A Circulating pool- present in large and present in large and

small blood vesselssmall blood vesselsA Marginating pool- A Marginating pool- that is arrested in that is arrested in

capillaries.capillaries.

Margination - regulated by selectin-Margination - regulated by selectin-mediated capture from the mediated capture from the bloodstream. bloodstream.

RollingRolling

Rolling adhesion of neutrophils to the Rolling adhesion of neutrophils to the endothelium is mediated by endothelium is mediated by L-selectinL-selectin on the neutrophil and on the neutrophil and P- and E-selectin P- and E-selectin on the endothelium. on the endothelium.

RollingRolling

Rolling allows interaction between CXC Rolling allows interaction between CXC chemokines such as:chemokines such as:

IL-8(Endothelial Cell’s surface)

β2 integrin expressions

AdhesionAdhesion

P, L-selectins &CD44

(Neutrophils)

P, E-selectins (Endothelial Cells)

Firm adhesion to the endothelium

LFA-1: Leukocyte function-ass’d Ag; Mac-1: Macrophage 1-Ag

The βThe β22 integrin’s 4 different integrin’s 4 different

heterodimers: heterodimers: 1. CD11a/CD18 or LFA-11. CD11a/CD18 or LFA-1 2. CD11b/CD18 or Mac-1; 2. CD11b/CD18 or Mac-1; 3. CD11c/CD18 or p150,953. CD11c/CD18 or p150,95 4. CD11d/CD18. 4. CD11d/CD18.

DiapedesisDiapedesis

Transmigration of Neutrophils from the Transmigration of Neutrophils from the intravascular compartment to the site of intravascular compartment to the site of infection by deformation and elongation. infection by deformation and elongation.

• Transendothelial migration of neutrophils

Diapedesis: Diapedesis: Endothelial cell interactions

PARACELLULAR(between endothelial cells)

• Transendothelial migration of neutrophils

Diapedesis:Diapedesis: Endothelial cell interactions

Transcellular (directly through endothelial

cells)

Transmigratory cups highICAM-1 VCAM-1

Diapedesis: Diapedesis: Epithelial cell interactions This process involves three stages:This process involves three stages:

Epithelial adhesion Epithelial adhesion

MigrationMigration

Post-MigrationPost-Migration

Cross Section of Blood VesselsMARGINATION

Epithelial adhesion Epithelial adhesion

Neutrophil Transmigration

MigrationMigration

Neutrophil transmigrateAcross

Epithelial cell wall

MigrationMigration

ChemotaxisChemotaxis

Once through the endothelial basement Once through the endothelial basement membrane, neutrophils migrate along a membrane, neutrophils migrate along a chemotactic gradient. chemotactic gradient.

Neutrophil chemotactic proteins include Neutrophil chemotactic proteins include Chemokines (e.g., IL-8)Chemokines (e.g., IL-8) Complement split products (e.g., C5a)Complement split products (e.g., C5a) Bacterial products (e.g., N-formyl methionyl Bacterial products (e.g., N-formyl methionyl

peptides), peptides), Lipid mediators (e.g., LTBLipid mediators (e.g., LTB44) )

• IL-8- IL-8- produced by macrophages, epithelial produced by macrophages, epithelial cells and neutrophils.cells and neutrophils.

• IL-8 is a very strong chemoattractant for IL-8 is a very strong chemoattractant for neutrophils and T-lymphocytes.neutrophils and T-lymphocytes.

Chemotaxis: Chemokine (Endogenous Factor)

Chemotaxis: Functions of Complement (Endogenous Factor)

• C5aC5a (C3a, C4a) (C3a, C4a) act on specific receptors to act on specific receptors to produce similar local inflammatory responses produce similar local inflammatory responses (anaphylatoxins). (anaphylatoxins).

• All three induce smooth muscle contraction All three induce smooth muscle contraction and increase vascular permeability.and increase vascular permeability.

Chemotaxis: Chemokine (Endogenous Factor)

• C5aC5a also acts directly on neutrophils also acts directly on neutrophils to increase their adherence to vessel to increase their adherence to vessel walls, their migration toward sites of walls, their migration toward sites of antigen deposition, and their ability to antigen deposition, and their ability to ingest particles.ingest particles.

Chemotaxis: Chemokine (Endogenous Factor)

1.1. N-formylated oligopeptides (FMLP)N-formylated oligopeptides (FMLP)

2.2. Endotoxin/Lipopolyssacharide (LPS)Endotoxin/Lipopolyssacharide (LPS)

Chemotaxis: Bacterial Products (Exogenous Factor)

LPS- (endotoxin)

Chemotaxis: Bacterial Products (Exogenous Factor)

LBP

LPS-LBP Complex

↑CD11b/CD18High

ADHESIVE activity

PhagocytosisPhagocytosis

PhagocytosisPhagocytosis

Degranulation:Degranulation:Neutrophil enzymesNeutrophil enzymes

• Azurophilic or PrimaryAzurophilic or Primary• These are the first granules formed in the These are the first granules formed in the

developing neutrophil (Promyelocyte)developing neutrophil (Promyelocyte)

• Specific or SecondarySpecific or Secondary• These granules are formed later in the These granules are formed later in the

development of the neutrophil (myelocyte) development of the neutrophil (myelocyte)

Neutrophil granule contentsNeutrophil granule contents

Neutrophil granule contentsNeutrophil granule contents

MMP-9: Matrix Metalloprotease

Primary GranulesPrimary GranulesMyeloperoxidase (MPO)ElastaseDefensinsLysozyme

OthersBPICathepsin GAlkaline phosphataseProteinase 3β-glucuronidaseα-fucosidasePhospholipases A2, C, Dα-mannosidase

Neutrophil enzymesNeutrophil enzymes

Neutrophil enzymesNeutrophil enzymes

• Myeloperoxidase (MPO)Myeloperoxidase (MPO): : is an abundant is an abundant granular enzyme (accounts for 5% of dry granular enzyme (accounts for 5% of dry weight of the neutrophil). weight of the neutrophil).

• This enzyme combines hydrogen This enzyme combines hydrogen peroxide with chloride ions to form peroxide with chloride ions to form hypochlorous acid (HOCl = bleach).hypochlorous acid (HOCl = bleach).

Secondary granulesLysozymeCollagenaseLactoferrin OthersGelatinaseVitamin B12-binding proteinCytochrome b558fMLP receptorCD11b/CD18, CD11c/CD18 (integrins)Complement receptor 3 (CR3)HistaminasePlasminogen activator

Neutrophil enzymesNeutrophil enzymes

Neutrophil enzymesNeutrophil enzymes

• LysozymeLysozyme: : like MPO, is a microbicidal like MPO, is a microbicidal enzyme. enzyme.

• LysozymeLysozyme digests debris from cell walls digests debris from cell walls of bacteria that have already been of bacteria that have already been processed by other enzymes. processed by other enzymes.

• Another function of Another function of lysozyme is to is to modulate inflammation by modulate inflammation by suppressingsuppressing neutrophil chemotaxis and oxidative neutrophil chemotaxis and oxidative metabolism.metabolism.

Within 30 seconds after a neutrophil ingests a particle, it begins to secrete specific granule components into the phagosome via phagolysosomal fusion.

Within 3 minutes, azurophil granule components are discharged into the phagolysosome.

2O2 2O2_

Heme

FAD

2e_

Extracellular space or

phagosome

CytoplasmNADPH

Oxidative Killing:Oxidative Killing: Superoxide-generating systemSuperoxide-generating system

Oxidative KillingOxidative Killing

HOCl: Hypoclorous acid; MPO: myeloperoxidases

NETosisNETosis

Neutrophil Extracellular Traps (NET)Neutrophil Extracellular Traps (NET)Composed of DNA and histonesComposed of DNA and histonesAntimicrobial proteins from its granulesAntimicrobial proteins from its granules

- MyeloperoxidesMyeloperoxides- ElastaseElastase- DefensinsDefensins- Protienase 3Protienase 3- Cathepsin GCathepsin G- CalprotectinCalprotectin

NETosisNETosis

Distinctive form of cell death Distinctive form of cell death - Disintegration of the nuclear envelopeDisintegration of the nuclear envelope- Mixing of the NET components and granules Mixing of the NET components and granules

contentscontents

- Occurs when cell membrane ruptures and Occurs when cell membrane ruptures and cell dies.cell dies.

- Upto 4 hours after activationUpto 4 hours after activation

NETs: NETs: Neutrophil Extracellular TrapsNeutrophil Extracellular Traps

Bacteria Bacteria trapped in trapped in

NETsNETs

Staph aureus

E. coli

Clearance & DeathClearance & Death

Three Mechanisms:Three Mechanisms:1.1. NETosisNETosis

2.2. ApoptosisApoptosis- Chromatin condensationChromatin condensation- Nuclear collapseNuclear collapse- Cytosolic vacuolationCytosolic vacuolation- Cell shrinkageCell shrinkage

1.1. NecrosisNecrosis

- cell burst and release of toxic contents- cell burst and release of toxic contents

> Maintain neutrophil number in the bloodRemoval of invadersResolution of inflammation

Inflammatory stimuli Macrophages Corticosteroid

• LungLung Adult Respiratory Distress SyndromeAdult Respiratory Distress Syndrome

AsthmaAsthma

AsbestosisAsbestosis

EmphysemaEmphysema

Idiopathic pulmonary fibrosisIdiopathic pulmonary fibrosis

Neutrophil-associated Neutrophil-associated diseasesdiseases

PULMONARY TRACTPULMONARY TRACTMarginating pool(20-60x higher)

DiapedesisTravel tru the Pulmonary capillary

Increase transit TIME

Increase concentration of Neutrophils

Exposure to:InhalantsCigaretteinfections

Decrease transit TIME(Bone Morrow)

Release of ImmatureNeutrophils to the Blood

Vast network of Capillary beds

Smaller vessel Diameter

Neutrophilic Neutrophilic AsthmaAsthma

• Mucus hypersecretion

• Impaired efferocytosis

• Bacterial persistence

- Symptomatic asthma and Symptomatic asthma and airway hyper- airway hyper- responsiveness in the responsiveness in the presence of a neutrophilic presence of a neutrophilic bronchitis, with sputum bronchitis, with sputum neutrophil counts > 61%.neutrophil counts > 61%.

- 10-30% of cases of stable 10-30% of cases of stable asthma in adultsasthma in adults

Immunomodulators of neutrophils functionsImmunomodulators of neutrophils functions

AP-1, activator protein-1; NF-B, nuclear factor-B; TNF, tumour necrosis factor-alpha; IL-8, interleukin-8.

AP-1:activator protein 1; CaMK:calmodulin kinase; DAG:diacylglycerol; EGFR:epidermal growth factor receptor; ERK:extracellular signal-regulated kinase; GFR:growth factor receptor; GPCR:G-protein-coupled receptor; IKK:IB kinase; IP3R:inositol triphosphate receptor; IRAK:IL-1 receptor-associated kinase; PKC:protein kinase C; TAK1:transforming growth factor-activated protein kinase

Other Options for Neutrophilic Other Options for Neutrophilic AsthmaAsthma

1.1. Reduced inhaled CS Reduced inhaled CS

2.2. Bacterial eradicationBacterial eradication

3.3. Anti- IL-8 antibodyAnti- IL-8 antibody

4.4. Anti alpha1 antitrypsin antibodyAnti alpha1 antitrypsin antibody

5.5. PPAR agonistPPAR agonist

6.6. TheophyllineTheophylline

7.7. CXCR2 AntagonistCXCR2 Antagonist

Summary:Summary:

Discussed the neutrophils’ biologyDiscussed the neutrophils’ biology The Birth The Birth The MorphologyThe Morphology

Discussed each step of its Key Role in innate Discussed each step of its Key Role in innate immune defenses (Phagocytosis, ROI, immune defenses (Phagocytosis, ROI, NETosis)NETosis)

Neutrophil-Associated Diseases (N. Asthma)Neutrophil-Associated Diseases (N. Asthma) Discuss some Immunomodulators of Discuss some Immunomodulators of

neutrophils functions (macrolide)neutrophils functions (macrolide)